ABSTRACT The increasing use of soy-based products in the diet has raised concerns regarding their reproductive toxicity because soybeans contain isoflavones which exhibit hormonal activity and may interfere with reproductive development. The predominant isoflavones in soybeans, genistin and daidzin, are hydrolyzed in the gastrointestinal tract to genistein and daidzein aglycones, and daidzein is further broken down to equol. Of interest, the male phenotype is supported primarily by the sex steroid testosterone which is produced by testicular Leydig cells. In this application, Aim 1 experiments will test the hypothesis that timing of exposure influences the hormonal effects of soy-based diets in the testis. Male rats will be fed either a soy-free control or soy-based diet at different times in the postnatal period followed by analysis of serum hormone levels and correlation of the timing of exposure to the impact on Leydig and germ cell development. In Aim 2, PI hypothesizes that genistein, daidzein and equol all contribute to isoflavone exposure effects. Weanling male rats will be fed either the control, soy-based diet, or the control diet supplemented with genistin, daidzin or equol from 21-35 days of age. To determine if isoflavones interfered with steroid hormone regulation of the hypothalamus and pituitary gland, cohorts of control and isoflavone-exposed animals will be castrated and implanted with silastic capsules containing testosterone, dihydrotestosterone (a non-aromatizable androgen) or 17?-estradiol (the natural estrogen) from days 36-56 postpartum. Samples will be obtained for immunohistochemical analysis of steroid hormone receptor [estrogen receptors (ESR1, ESR2) and androgen receptor (AR) protein in the preoptic area and ventromedial nucleus of the hypothalamus. In addition, gonadotropin releasing hormone (GnRH) receptor, steroid hormone receptors and gonadotropin (luteinizing and follicle stimulating hormone) subunit (LH? and FSH?) expression will be analyzed in western blots of pituitary glands. In the third aim, experiments will be performed to determine the molecular basis for organizational (permanent) effects of isoflavones in Leydig cells. Timed-pregnant dams will be maintained either on the control or soy-based diet during the period of primordial germ cell migration, i.e., gestational days 7-21. With male offspring as the F1 generation, mating studies will be performed to generate F2 and F3 generations of male rats. Mesenchymal stem cells and fully differentiated adult Leydig cells, obtained from 7- and 90-day old male rats, will be evaluated for developmental potential and terminal differentiation. In addition, genomic DNA and total RNA will be analyzed for DNA methylation patterns and microRNA expression. Altogether, results will describe how isoflavone effects impact Leydig cell development and paracrine regulation of testicular cells, interfere with steroid hormone regulation of hypothalamic and pituitary gland function, and identify epigenetic mechanisms which perpetuate isoflavone exposure effects across generations. These data will advance mission of the National Institute of Environmental Health Sciences to discover how the environment affects people in order to promote healthier lives. 1